Thrombospondin (TSP)-1 is an approximately 420kDa trimeric glycoprotein that is secreted by numerous host tissues including the endothelial cell (EC). This multidomain molecule recognizes several EC receptors and induces multiple and sometimes conflicting signaling events and biological responses. That EC can both produce and respond to TSP-1 suggests autocrine/ paracrine regulation. TSP-1 includes EC attachment to and spreading on substrates, actin organization, cell motility, and angiogenesis. Recently we have demonstrated that TSP-1 opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine phosphorylation. The zonula adherens (ZA) is an intercellular adherens junction that is physically coupled to the actin cytoskeleton and regulated through protein tyrosine phosphorylation. In EC, a group of cytoplasmic proteins, collectively termed catenins, form a multiprotein complex that tethers actin to the cytoplasmic domain of vascular endothelial (VE)-cadherin. the Cadherins are surface receptors that mediate homophilic intercellular adhesion. We now have demonstrated that TSP-1 induces tyrosine phosphorylation of two ZA components, gamma-catenin and p120Cas. To extend our findings, we propose the following Specific Aims: 1. To determine whether the interaction(s) Of 2 specific sequence(s) within the TSP-1 interacts with an EC receptor(s) that is coupled to tyrosine phosphorylation events and opening of the endothelial paracellular pathway. 2. To determine whether TSP-1 opens the endothelial paracellular pathway through tyrosine phosphorylation-dependent ZA disassembly. 3. To determine whether TSP-1 opens the endothelial paracellular pathway through tyrosine phosphorylation-dependent actin reorganization and/or disruption of the ZA-actin cytoskeletal linkage. The proposed studies are designed to identify the TSP-1 sequence(s), the EC receptor, and the tyrosine phosphorylation events through which TSP-1 perturbs protein-protein interactions promoting ZA disassembly, actin reorganization, and/or disruption of ZA-actin cytoskeletal linkage, and finally, opening of the endothelial paracellular pathway.